A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade

Irene Izquierdo; María N. Barrachina; Lidia Hermida-Nogueira; Vanessa Casas; Luis A. Morán; Serena Lacerenza; Roberto Pinto-Llorente; Johannes A. Eble; Vivian de los Ríos; Eduardo Domínguez; María I. Loza; José Ignacio Casal; Montserrat Carrascal; Joaquín Abián; Angel García

doi:10.1055/s-0039-3400295

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2020; 120(02): 262-276
DOI: 10.1055/s-0039-3400295

Cellular Haemostasis and Platelets

Georg Thieme Verlag KG Stuttgart · New York

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade

Irene Izquierdo

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

,

María N. Barrachina

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

,

Lidia Hermida-Nogueira

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

,

Vanessa Casas

²CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain

,

Luis A. Morán

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

,

Serena Lacerenza

³Department of Pharmacy, University of Pisa, Pisa, Italy

,

Roberto Pinto-Llorente

²CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain

,

Johannes A. Eble

⁴Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany

,

Vivian de los Ríos

⁵Proteomics Facility, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain

,

Eduardo Domínguez

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

,

María I. Loza

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

,

José Ignacio Casal

⁵Proteomics Facility, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain

,

Montserrat Carrascal

²CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain

,

Joaquín Abián

²CSIC/UAB Proteomics Laboratory, Institute of Biomedical Research of Barcelona, Spanish National Research Council (IIBB-CSIC/IDIBAPS), Barcelona, Spain

,

Angel García

¹Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidad de Santiago de Compostela, and Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain

› Author Affiliations

Abstract

C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A₂ (TXA₂). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA₂ at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr^525+526) and Src(p-Tyr⁴¹⁹), and for PLCγ2 activity (p-Tyr⁷⁵⁹). We demonstrated that Syk phosphorylation at Tyr^525+526 also happens in the presence of ADP and TXA₂ inhibitors, which is not the case for Src-pTyr⁴¹⁹ and PLCγ2-pTyr⁷⁵⁹. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca²⁺ mobilization assays confirmed the relevance of ADP and TXA₂ for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome.

Keywords

platelets - CLEC-2 signaling - tyrosine phosphoproteome

Full Text

References

References
1 Watson SP, Herbert JM, Pollitt AY. GPVI and CLEC-2 in hemostasis and vascular integrity. J Thromb Haemost 2010; 8 (07) 1456-1467
2 Suzuki-Inoue K, Inoue O, Ding G. , et al. Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets. J Biol Chem 2010; 285 (32) 24494-24507
3 Suzuki-Inoue K. Roles of the CLEC-2-podoplanin interaction in tumor progression. Platelets 2018; 1-7
4 Hughes CE, Pollitt AY, Mori J. , et al. CLEC-2 activates Syk through dimerization. Blood 2010; 115 (14) 2947-2955
5 Suzuki-Inoue K, Fuller GL, García A. , et al. A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2. Blood 2006; 107 (02) 542-549
6 Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK, Kato Y, Yamaguchi Y. A platform of C-type lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin and nonglycosylated rhodocytin. Structure 2014; 22 (12) 1711-1721
7 Gross BS, Melford SK, Watson SP. Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets. Eur J Biochem 1999; 263 (03) 612-623
8 Gross BS, Lee JR, Clements JL. , et al. Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets. J Biol Chem 1999; 274 (09) 5963-5971
9 Pasquet JM, Gross B, Quek L. , et al. LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI. Mol Cell Biol 1999; 19 (12) 8326-8334
10 Manne BK, Badolia R, Dangelmaier C. , et al. Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets. J Biol Chem 2015; 290 (18) 11557-11568
11 Hughes CE, Sinha U, Pandey A, Eble JA, O'Callaghan CA, Watson SP. Critical Role for an acidic amino acid region in platelet signaling by the HemITAM (hemi-immunoreceptor tyrosine-based activation motif) containing receptor CLEC-2 (C-type lectin receptor-2). J Biol Chem 2013; 288 (07) 5127-5135
12 Pollitt AY, Grygielska B, Leblond B, Désiré L, Eble JA, Watson SP. Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac. Blood 2010; 115 (14) 2938-2946
13 Badolia R, Inamdar V, Manne BK, Dangelmaier C, Eble JA, Kunapuli SP. G_q pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets. J Biol Chem 2017; 292 (35) 14516-14531
14 Parguiña AF, Alonso J, Rosa I. , et al. A detailed proteomic analysis of rhodocytin-activated platelets reveals novel clues on the CLEC-2 signalosome: implications for CLEC-2 signaling regulation. Blood 2012; 120 (26) e117-e126
15 García A. Two-dimensional gel electrophoresis in platelet proteomics research. Methods Mol Med 2007; 139: 339-353
16 Casanovas A, Pinto-Llorente R, Carrascal M, Abian J. Large-scale filter-aided sample preparation method for the analysis of the ubiquitinome. Anal Chem 2017; 89 (07) 3840-3846
17 Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 2015; 43 (Database issue): D512-D520
18 Szklarczyk D, Morris JH, Cook H. , et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 2017; 45 (D1): D362-D368
19 Raaijmakers LM, Giansanti P, Possik PA. , et al. PhosphoPath: visualization of phosphosite-centric dynamics in temporal molecular networks. J Proteome Res 2015; 14 (10) 4332-4341
20 Shannon P, Markiel A, Ozier O. , et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003; 13 (11) 2498-2504
21 UniProt Consortium T. UniProt: the universal protein knowledgebase. Nucleic Acids Res 2018; 46 (05) 2699
22 Zhang W, Trible RP, Zhu M, Liu SK, McGlade CJ, Samelson LE. Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell antigen receptor-mediated signaling. J Biol Chem 2000; 275 (30) 23355-23361
23 Tsang E, Giannetti AM, Shaw D. , et al. Molecular mechanism of the Syk activation switch. J Biol Chem 2008; 283 (47) 32650-32659
24 Zhang Z, Shen K, Lu W, Cole PA. The role of C-terminal tyrosine phosphorylation in the regulation of SHP-1 explored via expressed protein ligation. J Biol Chem 2003; 278 (07) 4668-4674
25 Spring K, Chabot C, Langlois S. , et al. Tyrosine phosphorylation of DEP-1/CD148 as a mechanism controlling Src kinase activation, endothelial cell permeability, invasion, and capillary formation. Blood 2012; 120 (13) 2745-2756
26 Senis YA, Antrobus R, Severin S. , et al. Proteomic analysis of integrin alphaIIbbeta3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions. J Thromb Haemost 2009; 7 (10) 1718-1726
27 Krause M, Sechi AS, Konradt M, Monner D, Gertler FB, Wehland J. Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton. J Cell Biol 2000; 149 (01) 181-194
28 Obergfell A, Judd BA, del Pozo MA, Schwartz MA, Koretzky GA, Shattil SJ. The molecular adapter SLP-76 relays signals from platelet integrin alphaIIbbeta3 to the actin cytoskeleton. J Biol Chem 2001; 276 (08) 5916-5923
29 Deneka A, Korobeynikov V, Golemis EA. Embryonal Fyn-associated substrate (EFS) and CASS4: the lesser-known CAS protein family members. Gene 2015; 570 (01) 25-35
30 Carrim N, Walsh TG, Consonni A, Torti M, Berndt MC, Metharom P. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation. PLoS One 2014; 9 (11) e113679
31 Séverin S, Nash CA, Mori J. , et al. Distinct and overlapping functional roles of Src family kinases in mouse platelets. J Thromb Haemost 2012; 10 (08) 1631-1645
32 Coxon CH, Geer MJ, Senis YA. ITIM receptors: more than just inhibitors of platelet activation. Blood 2017; 129 (26) 3407-3418
33 Mori J, Pearce AC, Spalton JC. , et al. G6b-B inhibits constitutive and agonist-induced signaling by glycoprotein VI and CLEC-2. J Biol Chem 2008; 283 (51) 35419-35427
34 Mazharian A, Wang YJ, Mori J. , et al. Mice lacking the ITIM-containing receptor G6b-B exhibit macrothrombocytopenia and aberrant platelet function. Sci Signal 2012; 5 (248) ra78
35 Ming Z, Hu Y, Xiang J, Polewski P, Newman PJ, Newman DK. Lyn and PECAM-1 function as interdependent inhibitors of platelet aggregation. Blood 2011; 117 (14) 3903-3906
36 Cicmil M, Thomas JM, Leduc M, Bon C, Gibbins JM. Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets. Blood 2002; 99 (01) 137-144
37 Newman PJ, Newman DK. Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology. Arterioscler Thromb Vasc Biol 2003; 23 (06) 953-964
38 Moraes LA, Barrett NE, Jones CI. , et al. Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells. J Thromb Haemost 2010; 8 (11) 2530-2541
39 Barrow AD, Astoul E, Floto A. , et al. Cutting edge: TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2. J Immunol 2004; 172 (10) 5838-5842
40 Naik MU, Caplan JL, Naik UP. Junctional adhesion molecule-A suppresses platelet integrin αIIbβ3 signaling by recruiting Csk to the integrin-c-Src complex. Blood 2014; 123 (09) 1393-1402
41 Mori J, Nagy Z, Di Nunzio G. , et al. Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148. Blood 2018; 131 (10) 1122-1144
42 Senis YA, Tomlinson MG, Ellison S. , et al. The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis. Blood 2009; 113 (20) 4942-4954
43 Mazharian A, Mori J, Wang YJ. , et al. Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function. Blood 2013; 121 (20) 4205-4220
44 Mori J, Wang YJ, Ellison S. , et al. Dominant role of the protein-tyrosine phosphatase CD148 in regulating platelet activation relative to protein-tyrosine phosphatase-1B. Arterioscler Thromb Vasc Biol 2012; 32 (12) 2956-2965
45 Durrant TN, Hutchinson JL, Heesom KJ. , et al. In-depth PtdIns(3,4,5)P₃ signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function. Blood Adv 2017; 1 (14) 918-932
46 Marshall AJ, Niiro H, Lerner CG. , et al. A novel B lymphocyte-associated adaptor protein, Bam32, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase. J Exp Med 2000; 191 (08) 1319-1332
47 Allam A, Niiro H, Clark EA, Marshall AJ. The adaptor protein Bam32 regulates Rac1 activation and actin remodeling through a phosphorylation-dependent mechanism. J Biol Chem 2004; 279 (38) 39775-39782

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